In a previous study, our laboratory reported increased CD38 expression in HIVE brains, which co-localized with astrocytes in areas of inflammation . The study established an important role for CD38 in modulating astrocyte neuroinflammatory responses. Here, we extend our analyses by investigating molecular mechanisms and signaling pathways responsible for CD38 modulation in astrocytes. In the present study, we show a direct upregulation of astrocyte-CD38 mediated by HIV-1. Transfection of astrocytes with HIV-1YU-2 gene expression plasmid not only increased CD38 mRNA and protein levels but also led to activation of astrocytes, as evident by an increase in production of chemokines CXCL8 and CCL2. In vivo, the increased CCL2 is thought to assist in attracting monocytes across the blood brain barrier. It is also implicated that proinflammatory chemokine CCL2 appears in brain soon after the virus enters the CNS . The results suggest that chemokines produced by a limited number of infected astrocytes may lead to immune cell recruitment and subsequent activation of non-infected astrocytes, thereby further upregulating astrocyte-CD38 as a whole. As we previously reported, increased CD38 enzyme activity leads to increased cADPR levels and a corresponding rise in intracellular calcium flux in activated astrocytes [14, 18]. The CD38/cADPR system is thought to initiate astrocyte to neuron calcium signaling, which then leads to increased release of neuromodulators from glial cells . Imbalance in calcium signaling may eventually lead to neuronal dysfunction .
Astrocytes may not be capable of de novo viral replication, but HIV-1-infected astrocytes can transmit the virus to CD4+ cells. Viral particles are released from astrocytes without reverse transcription. While this mode of infection does not increase viral load; it can, however, lead to viral persistence and spreading throughout the CNS [49, 50]. Since astrogliosis is a prominent feature of early CNS HIV-1 infection [51, 52], astrocytes are likely to be neuroprotective at the early phase of infection. However, dysfunction of astrocytes during chronic HIV-1 CNS infection and immune activation may lead to neurotoxicity [5, 39, 53]. The precise functional consequences of astrocyte infection and/or activation by HIV-1 remain unclear. Thus, using the model system of transfecting astrocytes with HIV-1 plasmid, we may be able to understand the direct effects of the viral gene expression on astrocyte function and their final impact on neurotoxicity during HIV-1-CNS infection.
Increased IL-1β expression has not been reported in astrocytes in response to various HIV-1 proteins or HIV-1 gene expression and replication models [54–56]. However, IL-1β is elevated in the brain tissues of patients infected with HIV-1 , is upregulated and secreted by infected/activated immune cells in the proinflammatory setting of HIV-1 infection , and induction of the IL-1β autocrine loop leads to further production of IL-1β and other cytokines . IL-1β along with TNF-α is also known to reactivate latent or non-production HIV-1 infection of astrocytes  in an NF-κB dependent manner . Therefore, subsequent signaling studies were performed in the context of IL-1β-induced CD38 expression.
We evaluated the role of transcription factor NF-κB in CD38 regulation. Our study showed that pretreatment of the astrocytes with SN50, a cell permeable peptide inhibitor of NF-κB, blocked the expected CD38 upregulation seen upon IL-1β-activation. This finding strongly emphasized that IL-1β-mediated gene upregulation involved the transcription factor NF-κB. This was further supported by attenuated CD38 expression and enzyme activity following transient transfection of astrocytes with IκBαM, which impeded NF-κB activation. Understanding the regulation of this signaling pathway during neuroinflammatory conditions like HIVE may have important therapeutic implications. The transcription factor NF-κB is a crucial mediator in the IL-1β signaling pathway and acts as a major driving force behind the induction of cytokines, chemokines and adhesion molecules by astrocytes; also important mediators of inflammation during HIVE . Following stimulation, the duration of NF-κB activation may be transient or persistent, depending on the cellular stimulus and cell type. Interestingly, it has been shown that stimulation with IL-1β may result in prolonged NF-κB activation, thus suggesting its implication in neuroinflammation associated with HIVE . Thus, taken together, these findings suggest that NF-κB is one of the major regulators of CD38 expression and enzyme activity in activated astrocytes.
We also investigated the involvement of MAPK in CD38 regulation, since NF-κB is downstream transcription factor in MAPK signaling cascade. Emerging evidence suggests that MAPK signaling pathway may play an important role in activated glia-induced neuronal malfunction . MAPKs are important in the transduction of extracellular signals into cellular responses. When activated, these kinases can phosphorylate both cytosolic and nuclear target proteins resulting in the activation of transcription factors and ultimately the regulation of gene expression . IL-1β is known to increase the activation of p38Ks, JNK and ERK MAPKs in primary astrocytes [26, 44]. We inhibited the activation of each MAPK pathway independently and showed significant decreases in CD38 expression in IL-1β-activated astrocytes. The IL-1β-induced ADP-ribosyl cyclase activity of CD38 was also significantly reduced by inhibition of each of the p38Ks, JNK and ERK pathways. It should be noted that inhibition of each individual signaling pathway alone, produced robust downregulation in CD38 expression and cyclase activity in IL-1β-activated astrocytes. It is therefore reasonable to assume equal importance of all three MAPK pathways in CD38 regulation. Importantly, the MAPK inhibitors did not affect basal CD38 levels in non-activated astrocytes. Thus, taken together these results suggest that MAPKs regulate IL-1β-induced CD38 levels in astrocytes, either directly or indirectly, through NF-κB. Both p38Ks and JNK have been reported to mediate neuronal damage primarily by glial activation . The activation of p38Ks plays an important role in developing HIV-1 envelope protein gp120-mediated cytotoxicity of human brain microvascular endothelial cells . MAPK activation can lead to nitric oxide production and cytokine release in glial cells, thus exacerbating the neuroinflammatory milieu during neurodegenerative disorders including HIVE [63, 64].
It is known that HIV-1 can activate p38Ks, ERK and JNK MAPK cascades, while HIV-1-transactivator may induce both NF-κB and p38Ks, JNK MAPK pathways [65, 66] in astrocytes. This may eventually lead to release of glutamate and pro-inflammatory cytokines from glial cells, thus contributing to neurodegeneration during HAD . HIV-1gp120 may also activate MAPKs in neurons . Activation of the NF-κB and MAPK signaling may lead to activation of nitric oxide synthase which can result in release of nitric oxide in both human and rat astrocytes and in C6 glioma cells [69, 70]. It has been reported previously that NF-κB activation may lead to release of reactive oxygen species, which in turn regulate inducible nitric oxide synthase expression in astrocytes (as reviewed in ). Thus, it will be interesting to understand how modulation of CD38 participates in the release of inducible nitric oxide synthase in IL-1β-activated astrocytes.
It is now well established that activated astrocytes release several inflammatory cytokines and chemokines including IL-1β, IL-6, TNF-α, CCL2 and CXCL8 (reviewed in [72, 73]), which are thought to contribute to inflammation associated with HIVE . We have previously demonstrated that the proinflammatory cytokine IL-1β upregulates Fas ligand in astrocytes, which induces apoptosis in neurons [45, 53], and that IL-1β-mediated production of CCL2 and CXCL8 is partially regulated by CD38 . Autocrine production of IL-1β can enhance a number of other signaling molecules downstream of the IL-1β signaling cascade . However, we have also shown CD38 expression is independent of the IL-1β-autocrine loop in astrocytes . Therefore, regulation of CD38 in astrocytes is net effect of a complex mechanism.